Melanoma is a malignant skin cancer with few effective treatment options for advanced stages of the disease. It is important to develop effective treatment strategies that will eliminate existing tumors and/or prevent the formation or recurrence of tumors. The work proposed in this application is to use an immunotherapy approach in the form of a gene delivery system. Electroporation (EP) will be used to introduce a plasmid DNA encoding for a cytokine into melanoma cells promoting an immune response to reduce cancer growth and prevent development. Interleukin 15 (IL-15) is a cytokine that activates natural killer (NK) cells and stimulates CD8+ T cells to exhibit long-term memory CTL activity against target cells. The research in this study focuses on delivering plasmid IL-15 (plL-15) by EP into a mouse model injected with B16.F10 murine melanoma cells. In Specific Aim 1, we will evaluate IL-15 expression levels when applying different EP parameters and different plasmid concentrations. An ELISA and real time RT PCR analysis will be used to detect protein levels and mRNA levels produced by the tumor cells, respectively. In Specific Aim2, the high and low expression levels of IL-15 obtained from Specific Aim 1 will be used to correlate expression levels of IL-15 with tumor regression. We will analyze the local and systemic cellular infiltrate stimulated by plL-15/EP using FACS, immunohistochemistry, Luminex bead array, TUNEL and ELISPOT to understand the reduction of tumor growth. A depletion study using monclonal antibodies will be used to demonstrate the roles that innate NK cells and adative T cells play when they are separately depleted. The reaction and duration of the response by these cells in the absence of one another will be evaluated when stimulated by the production of IL-15 from tumor cells. ELISPOT and FACS analysis will be used to demonstrate the results from the depletion studies. In Specific Aim 3, we will also determine the extent of IL-15 expression levels that provide viable treatments for metastatic disease using a two-tumor model. The cellular systemic responses to intratumoral treatments will be analyzed using immunohistochemistry, ELISPOT and ELISA. The relavance of this research is that the administration of plL-15/EP will provide promising stimulation of the immune response against tumor cells without causing toxicity or deleterious effects to the surrounding tissues. These experiments can promote the development of a potential anti-cancer therapy to block cancer metastasis as well as the recurrences of new diseases. [unreadable] [unreadable] [unreadable]